In order for many drugs and imaging agents to have therapeutic potential it is necessary for them to be delivered to the proper location in the body. In many cases, agents are ineffective because it is impossible, with the available technology, to deliver them to the proper locations within a biological organism. Thus, a main focus in pharmaceutical research is in the delivery of agents to tissues and cells.
An area of development aggressively pursued by researchers is in the delivery of agents not only into a cell but into the cell's cytoplasm and further yet, into the nucleus. This area of research is being pursued in particular for delivery of biological agents such as DNA, RNA, ribozymes and proteins. Not only do these materials hold great promise as therapeutic agents, but some workers believe that in some diseases they may act as the "magic bullet" i.e., curing an illness without deleterious side effects. Examples of therapeutic pursuits include the application of antisense technology. Briefly, the strategy in antisense technology is to deliver an agent such as DNA oligonucleotide that binds to sites on messenger RNA (mRNA) which directs the production of proteins related to disease. Other strategies include the use of triplex agents which bind to the double helix to interfere with transcription, preventing the production of mRNA. In spite of these developments, numerous "delivery" problems have surfaced.
One major problem encountered is that effective delivery of oligonucleotides across cell membranes is difficult, if not impossible. Another problem facing workers is that oligonucleotides are degraded by intracellular and extracellular enzymes such as exonucleases and endonucleases. In order to overcome these problems, workers have modified the typical phosphodiester form of oligonucleotides. For example, modifications have included the creation of phosphorothioate and methylphosphonate oligonucleotides. Methylphosphonate oligonucleotides contain uncharged sectors which increase intracellular uptake and resist enzymatic degradation. Phosphonothioate oligonucleotides are negatively charged but are resistant to endonucleases. Although these modified oligonucleotides show promise as therapeutics, there is no evidence of in vivo success. Further, workers have not provided a delivery vehicle capable of introducing a sufficient quantity of oligonucleotides into the cell such that it has therapeutic effect. Since it is desirable to load a cell with s numerous oligonucleotides, workers have begun to focus their efforts on developing a delivery vehicle which not only acts to deliver an agent into the cytoplasm and nucleus of a cell, but also delivers oligonucleotides in large numbers.
Cationic liposomes have been used to introduce DNA into vascular cells. Plautz, G. E., et al, "Liposome Mediated Gene Transfer into Vascular Cells", J. Liposome Research 3(2):179-199 (1993); Felgner, P. L., et al. "Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure", Proc. Natl. Acad. Sci. 84:7413-7417 (1987). For example, Lipofectin, liposomes formed from a mixture of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE), has been used to transduce recombinant genes into coronary arteries in vivo. Another example of a cationic liposome used to introduce genes into DNA is a liposome preparation containing DC-chol, 3b[N-(9N'N'-dimethylaminoethane)-carbamoyl] cholesterol and DOPE. Other efforts have been directed toward the use of modified retroviruses and cationic liposomes for gene transfer.
Liposomes in general have been known. Liposomes are microscopic vesicles made from phospholipids, which form closed, fluid filled spheres when dispersed with aqueous solutions. Phospholipid molecules are polar, having a hydrophilic head and two hydrophobic tails consisting of long fatty acid chains. Thus, when a sufficient concentration of phospholipid molecules are present in aqueous solutions, the tails spontaneously associate to exclude water while the hydrophilic phosphate heads interact with water. The result is a spherical, bilayer membrane in which the fatty acid tails converge in the interior of the newly formed membrane, and the polar heads point in opposite directions toward an aqueous medium. These bilayer membranes thus form closed spheres, known as liposomes. The polar heads at the inner surface of the membrane point toward the aqueous interior of the liposome and, at the opposite surface of the spherical membrane, the polar heads interact with the surrounding aqueous medium. As the liposomes are formed, water soluble molecules can be incorporated into the aqueous interior, and lipophilic molecules may be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an aqueous center.
Methods for producing liposomes are well known in the art, and there are many types of liposome preparation techniques which may be employed to produce various types of liposomes. These can be selected depending on the use, the chemical intended to be entrapped, and the type of lipids used to form the bilayer membrane. The requirements which must be considered in producing a liposome preparation are similar to those of other controlled release mechanisms. They are: (1) a high percent of chemical entrapment; (2) increased chemical stability; (3) low drug toxicity; (4) rapid method of production; and (5) a reproducible size distribution.
The first method described to encapsulate drugs or other chemicals in liposomes involved the production of multilamellar vesicles (MLVs). Liposomes can also be formed as unilamellar vesicles (UVs), which generally have a size less than 0.5 .mu.m (.mu.m, also referred to as "microns"). There are several techniques known in the art which are used to produce unilamellar liposomes.
Smaller unilamellar vesicles can be formed using a variety of techniques, such as applying a force sufficient to reduce the size of the liposomes and or produce smaller unilamellar vesicles. Such force can be produced by a variety of methods, including homogenization, sonication or extrusion (through filters) of MLVs. These methods result in dispersions of UVs having diameters of up to 0.2 .mu.m, which appear as clear or translucent suspensions. Other standard methods for the formation of liposomes are know in the art, for example, methods for the commercial production of liposomes include the homogenization procedure described in U.S. Pat. No. 4,753,788 to Gamble, a preferred technique, and the method described in U.S. Pat. No. 4,935,171 to Bracken, which are incorporated herein by reference.
Another method of making unilamellar vesicles is to dissolve phospholipids in ethanol and injecting them into a buffer, whereby the lipids will spontaneously rearrange into unilamellar vesicles. This provides a simple method to produce UVs which have internal volumes similar to that of those produced by sonication (0.2-0.5 L/mol of lipid). Another common method for producing small UVs is the detergent removal technique. Phospholipids are solubilized in either ionic or non-ionic detergents such as cholates, Triton X-100, or n-alkylglucosides. The drug is then mixed with the solubilized lipid-detergent micelles. Detergent is then removed by one of several techniques: dialysis, gel filtration, affinity chromatography, centrifugation or ultrafiltration. The size distribution and entrapment efficiencies of the UVs produced this way will vary depending on the details of the technique used.
The therapeutic uses of liposomes include the delivery of drugs which are normally toxic in the free form. In the liposomal form the toxic drug may be directed away from the sensitive tissue and targeted to selected areas. Liposomes can also be used therapeutically to release drugs, over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming an aqueous dispersion of hydrophobic drugs for intravenous delivery.
When liposomes are used to target encapsulated drugs to selected host tissues, and away from sensitive tissues, several techniques can be employed. These procedures involve manipulating the size of the liposomes, their net surface charge as well as the route of administration. More specific manipulations have included labeling the liposomes with receptors or antibodies for particular sites in the body. The route of delivery of liposomes can also affect their distribution in the body. Passive delivery of liposomes involves the use of various routes of administration, e.g., intravenous, subcutaneous and topical. Each route produces differences in localization of the liposomes. Two methods used to actively direct the liposomes to selected target areas are binding either antibodies or specific receptor ligands to the surface of the liposomes. Antibodies are known to have a high specificity for their corresponding antigen and have been shown to be capable of being bound to the surface of liposomes, thus increasing the target specificity of the liposome encapsulated drug.
Since the chemical composition of many drugs precludes their intravenous administration, liposomes can be very useful in adapting these drugs for intravenous delivery. Furthermore, since liposomes are essentially hollow spheres made up of amphipathic molecules, they can entrap hydrophilic drugs in their aqueous interior space and hydrophobic molecules in their lipid bilayer. Unwanted molecules that remain in the dispersion external to the liposomes, such as unentrapped agents, are removed by column chromatography or ultrafiltration. Although methods for making liposomes are well known in the art, it is not always possible to determine a working formulation without experimentation.
U.S. Pat. No. 4,310,505, incorporated by reference, discloses lipid vesicle formulations containing amino sugar derivatives of cholesterol as cell-surface receptor analogs. The vesicles were found to release their contents in a controlled manner, and in some cases, to be rapidly concentrated in the lymphatic system and/or liver, lungs or spleen of the host. These liposomes were noted to be used in the treatment of lysosomal storage disease. Liposomes containing amino-sugar derivatives have been shown to localize in s aggregates of polymorphonuclear leukocytes. Mauk, M. R. et al., "Vesicle Targeting: Timed Release and Specificity for Leukocytes in Mice by Subcutaneous Injection" Science 207:309-311 (1980).
The delivery problems noted above become manifest in treating a disease such as arteriosclerosis, a disease associated with the hardening or narrowing of arterial walls and the leading cause of death in Western Society. The majority of these deaths is caused by atherosclerosis, a type of arteriosclerosis characterized by lipid deposits in the intima of large and medium size arteries. These deposits or lesions are typically classified as fatty streaks, fibrous plaques or complicated lesions. Fatty streaks, probably the earliest lesions of atherosclerosis, are generally characterized by a build-up of lipid-filled smooth muscle cells, macrophage and fibrous tissue in the intima. Advanced atherosclerosis gives rise to fibrous plaques, which are raised lesions and elevated areas of intimal thickening. These lesions consist of a central core of extracellular lipid and necrotic cell debris (gruel) covered by a fibromuscular layer containing large numbers of smooth muscle cells, macrophage, and collagen. The third class, known as the complicated lesion, is characterized by calcified fibrous plaques. Stenosis, or narrowing, of vascular passages can result from gradual occlusion as the plaques thicken and thrombi form.
Atherosclerosis and stenotic vascular lesions are typically treated through the use of percutaneous transluminal coronary angioplasty (PTCA) procedures. These methods, which include balloon angioplasty, the atherectomy catheter, the excimer laser, and the rotablator, are used to dilate the stenosed blood vessels. However, the efficacy of PTCA is limited by the development of restenosis, or renarrowing, of the treated area following PTCA due to neointima formation. Approximately 25% of successful first-time angioplasty procedures must be repeated within 6 months and an additional 10% of the patients must later undergo coronary bypass surgery because of restenosis. A discussion of the causative elements of restenosis appears in Bone, R. C., ed., "Restenosis after Coronary Angioplasty," Disease-A-Month, 39:616-670 (1993).
PTCA procedures often cause collateral injury to the arterial wall, which triggers a "healing" process involving a series of physiologic events that ultimately results in neointima formation. Neointima formation is a multi-step process involving a complex interaction among several growth factors which promote vascular smooth muscle cell (VSMC) proliferation and migration. In this process, erosion of the intima, the single continuous layer of endothelial cells lining the arterial walls, during angioplasty promotes platelet aggregation. The platelets adhere to the arterial wall and release growth factors (e.g., platelet-derived growth factor, thrombin, basic fibroblast growth factor and transforming growth factor) which initiate the migration of VSMC to the intima and promote the proliferation of VSMC comprising the tunica media. Growth factor-induced VSMC proliferation involves, in turn, a sequential activation of intracellular proteins which promote cell-cycle progression. The next step of the process is associated with an inflammatory response, evidenced by an invasion of inflammatory cells such as monocytes, macrophage, and other white blood cells. These inflammatory cells further induce and stimulate growth of VSMC in the media, which subsequently migrate into the intimal space and produce large amounts of extracellular proteoglytic matrix material, forming a neointima layer which represents the lesion. It appears that endothelium then grows over the injured area and restenosis results.
Considerable research has been undertaken to identify agents which prevent or reverse restenosis. For example, many agents have been examined in the prevention of restenosis. A comprehensive review of therapeutics potentially useful in the treatment of restenosis appears in Herrman, Jean-Paul R. et al. "Pharmacological Approaches to the Prevention of Restenosis Following Angioplasty, The Search for The Holy Grail?" (parts I and II) Drugs 46 (1): 18-52, 46 (2):249-262, 1993.
Other drugs considered for inhibiting restenosis include, but are not limited to, oligonucleotides (e.g., antisense gene therapy), protein kinase C, endothelial growth factor and anti-platelet activating agents. Efforts to prevent restenosis utilizing systemic administration of such drugs, however, have been hampered by generalized toxicity at effective dosages. Furthermore, successful therapy for preventing restenosis has been thwarted by the complexity of the physiological processes responsible for neointima formation as well as the inability to deliver effective quantities of drugs to the site of injury, due to inefficient cellular uptake and lack of specificity (i.e., targeting) to VSMC. One possible mechanism for the failure of drugs to provide effective prevention of restenosis is that the agents never reach the tissue targeted. For example, the candidate agents may simply be washed away from the treatment areas by blood flow.
Gene therapy, using antisense oligonucleotides which effectively "shut off" the genes involved in neointima formation, is a promising approach to the problem of restenosis. However, due to the multiplicity of growth factors involved in neointima formation, selective inhibition of any single growth factor is unlikely to completely prevent lesion formation. Accordingly, Morishita has shown inhibition of neointima hyperplasia in a rat carotid model following angioplasty injury by administration of two antisense oligonucleotides (AS-oligos) which block the cell-cycle regulatory genes for proliferating-cell nuclear antigen (PCNA) and p34.sup.cdc2 (cdc2). Morishita, R. et al. "Single intraluminal delivery of antisence cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia" Proc. Natl. Acad. Sci. 90:84774 (1993). PCNA, a nuclear protein required for DNA synthesis by DNA polymerase .DELTA., and cdc2, a serine/threonine protein kinase, are primary components of the cell-cycle progression which regulate VSMC proliferation. The AS-oligos were constructed to be directed to the translation initiation sites of PCNA and cdc2. Cellular uptake was enhanced and transfection stability of the AS-oligos increased by complexing the phosphorothioate AS-oligos with liposomes and the protein coat of inactivated Sendai hemagglutinating virus of Japan (HVJ). Morishita et al. observed more rapid uptake and a 10-fold increase in transfection efficiency of AS-oligos or plasmid DNA than standard lipofection or passive uptake methods. However, specific targeting of the HVJ AS-oligos liposome complex to VSMC was not reported. It is generally desired then to develop targeting specificity of drug carriers to VSMC since that would result in greater uptake by the smooth muscle cells at the site of injury and would permit the administration of higher, more effective dosages. Furthermore, it is advantageous to avoid the use of viral proteins in drug carriers as they present a potential biological hazard to humans.
Thus, it is an object of the present invention to provide a lipid construct that is superior and more effective in delivering agents into the cytoplasm and nucleus of a cell. It is yet another object of the present invention to provide for the targeting of an agent to vascular smooth muscle tissue. It is still yet a further object of this invention to provide for a less toxic and more efficacious treatment of such diseases as restenosis, viral diseases and cancer.